This application is for the continuance of R37GM54060, a Merit Award initially awarded as an ROI in 1994. The purpose of this grant is to determine how LPS recognition results in the syndrome of Gram-negative sepsis. The current cycle proposed four Specific Aims: 1) To characterize MD-2, 2) To characterize the fine details of TLR4/MD-2 receptor activation, 3) To characterize the TLR4 adapters: MyD88, Mai, TRIP and TRAM, 4) To define the biology of MD-2 in vivo. We have published 17 papers attributable to R37GM54060 so far in this funding cycle, and addressed most of the major issues in its Aims. We have reported on the opsonic activity of MD-2 and worked out the fine details of TLR4/MD-2 receptor activation. We have characterized important aspects of MyD88 and Mai interaction and function. We have definitively addressed the issue of altemative LPS receptors in macrophages. We have yet to generate a Mab to MD-2, although immunizations are in progress. We will launch our studies on the in vivo role of MD-2 in sepsis when mAbs are available. Several studies with respect to the adapter molecules are in progress. In the next 5 years, two new Aims are proposed. First, we plan to begin a new project designed to define the exact role of LPS in sepsis using a systems biology approach. We will begin by infecting a panel of knockout mice, each deficient in a major immune signaling pathway (e.g., MyD88, TRIP, Unc93b, ASC, etc) with a strain of Y pestis in which the acylation pattern of lipid A, as well as the bioactivity, is genetically controlled. LPS from a recombinant strain that is proinflammatory will also be tested vs. the far less inflammatory wild-type LPS. By examining gene expression and cytokine expression in these infected or LPS challenged mice, we will draw an INNATE IMMUNE RESPONSE MAP. Those pathways effected by LPS will be identified and separated from signaling pathways activated in infection independently of LPS. Finally, we will perform forward genetic screening on wild-derived inbred mice to identify new genes involved in LPS responses and use this to refine our Innate Immune Response Map. We believe that these studies will identify those aspects of septic shock that are amenable to anti-LPS therapy in order to help reduce the mortality associated with septic shock.